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PRE-B CELL RECEPTOR SIGNALING IN ACUTE LYMPHOBLASTIC
LEUKEMIA
by
Rahul R. Nahar
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
December 2010
Copyright 2010 Rahul R. Nahar

B cell lineage acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children and is also common in adults. In virtually all cases, B cell lineage ALL cells are arrested at the pre-B cell stage or before. ALL cases are categorized in different subgroups based on the different chromosomal translocations and abnormalities found in them. Philadelphia chromosome positive (Ph+) leukemia carrying the t(9;22)(q34;q11) translocation is defined by the constitutively active oncogenic tyrosine kinase BCR-ABL1. In addition, in >80% of cases deletions of IKAROS gene are found in Ph+ ALL. We have recently shown that Ph+ ALL can also be defined by deficiency of a functional pre-B cell receptor.; During normal B cell development, pre-B cell receptor signaling first induces proliferation of the B cell precursors (large cycling pre-B cells, Fraction C’) and then causes cell cycle arrest at the time of Ig light chain gene rearrangement to initiate differentiation (small pre-B cells, Fraction D). Here we show that expression of µ chain (immunoglobulin heavy chain) in BCR-ABL1 transformed cells or reconstitution of SLP65 (a major adaptor protein in pre-B cell receptor signaling complex) in Slp65-/- BCR-ABL1 cells induces cell cycle arrest and apoptosis.; Dissecting the mechanism of tumor suppression by the pre-B cell receptor in BCR-ABL1 ALL, we studied Stat5 signaling, a central mediator of survival and proliferation downstream of the BCR-ABL1 kinase. Forced expression of pre-B cell receptor or Ikaros resulted in rapid dephosphorylation of Stat5 Y694 and concomitant upregulation of BCL6. Upregulation of BCL6 is causally linked with pre-B cell receptor induced cell cycle arrest. By genome wide ChIP-on-chip analysis we observed direct recruitment of BCL6 transcriptional repressor to promoters of MYC and CCND2, two key mediators of cell cycle progression in Ph+ ALL. Forced expression of BCL6 induced a cell cycle arrest in BCR-ABL1 transformed ALL and in addition Bcl6-deficient BCR-ABL1 transformed pre-B cells were more resistant than the Bcl6+/+ cells to the tumor suppression by the pre-B cell receptor. Further overexpression of Myc could rescue the pre-B cell receptor mediated cell cycle arrest.; In the process of studying how the pre-B cell receptor results in dephosphorylation of Stat5, we found Ikaros as key mediator. We have recently shown that pre-B cell receptor signaling leads to upregulation of Ikaros. In addition, reconstitution of Ikaros resulted in significant Stat5 dephosphorylation and this event required the pre-B cell receptor associated adapter molecule SLP65. Further dominant negative Ikaros isoform IK6 prevents the cell cycle arrest induced by the pre-B cell receptor. As a consequence of Stat5 dephosphorylation Ikaros indirectly resulted in Bcl6 upregulation and subsequent cell cycle arrest.; BCR-ABL1 driven ALL is defined by deletions of Ikaros and absence of a functional pre-B cell receptor. However the mechanism of this tumor suppression by Ikaros/pre-B cell receptor still remained elusive. We here elucidate the mechanism of Ikaros mediated tumor suppressor, namely Stat5 dephosphorylation. In conclusion, we identify Stat5 dephosphorylation and BCL6 upregulation as the key events in the pre-B cell receptor / IKAROS mediated tumor suppression in Ph+ ALL.

PRE-B CELL RECEPTOR SIGNALING IN ACUTE LYMPHOBLASTIC
LEUKEMIA
by
Rahul R. Nahar
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
December 2010
Copyright 2010 Rahul R. Nahar